The long term objective of this project is to characterize the regulatory networks that control alternative pre-mRNA splicing in late erythropoiesis. The underlying hypothesis is that RNA processing is a critical regulator of gene expression, and that differentiation stage-specific splicing "switches" alter the structure and function of erythroid proteins as the cells are morphologically and functionally remodeled. In protein 4.1R pre-mRNA, exon 16 (E16) is skipped in early erythroid progenitors but included in later cells; this splicing switch is essential for spectrin-actin binding and red cell membrane mechanical stability. Preliminary studies have identified two additional splicing switches in these cells. Mechanistic studies with 4.1R pre-mRNA indicate that E16 is repressed in early erythroblasts due to binding of the splicing repressor, hnRNP A1, to silencer elements in E16; switching occurs via decreased A1 expression in late erythroblasts. In other systems, regulated alternative splicing is often mediated by "dynamic antagonism" between enhancer and silencer proteins binding competitively to regulatory sites in the RNA. Preliminary studies have identified enhancer elements in the 3' splice site region, the purine-rich region of E16, and the downstream intron. New data show that a novel RNA binding/splicing factor, Fox-2, acts at the intron enhancer to stimulate El6 splicing. To understand how multiple regulatory signals are integrated to determine E16 splicing, and to extend these studies to other erythroid genes, the following specific aims are proposed: (1) Explore E16 silencer and enhancer functions, focusing on enhancer identification and functional interactions with the A1 silencer. (2) Explore the mechanism of action of the intronic Fox-2 splicing enhancer, and its interactions with other E16 splicing regulators. (3) Explore the larger role of alternative splicing in erythropoiesis, by identifying/analyzing new erythroblast splicing switches besides the three currently known, and characterizing splicing factor expression patterns in erythroblasts. These aims will utilize RNA splicing assays and RNA:protein binding methods similar to those already applied to analysis of the A1 silencer, and will take advantage of recent computational and microarray technical advances. Achievement of these aims will increase our understanding of physiological splicing switches in erythroid cells, and provide new insights into function of novel Fox splicing enhancers of general importance to tissue-specific splicing in metazoan organisms. Many human diseases arise from genetic aberrations in pre-mRNA splicing, including defects in enhancer/silencer regulation; understanding the erythroid splicing program may thus have future therapeutic applications.